Isotopic labeling and isobaric labeling are two kinds of the typical quantification method that have been widely used in proteomics analysis. Herein, a novel quantitative strategy, partially isobaric peptide termini labeling (PITL), was developed to overcome the drawbacks of each above-mentioned labeling strategy, by simultaneously collecting the quantitative information from both MS and MS/MS spectrum. PITL is based on the site-selective N-terminus dimethylation labeling of peptide alpha-N-termini and the free epsilon-amino group of lysines, resulting in the partially isobaric labeling of peptides. The relative quantification can then be achieved by comparing the intensities of precursor ions in MS spectra and a, b and y ions in MS/MS spectra. The quantitative analysis of differently labeled yeast digests pooled with various ratios indicated the good quantitative accuracy, reproducibility, coverage and wide dynamic range of PITL strategy. Furthermore, PITL was applied to the quantitative proteome analysis of two mouse hepatocarcinoma ascites syngeneic cell lines with low and high lymph node metastasis rates (Hca-F and Hca-P). Given its low cost, simple operation and good accuracy, PITL might have great potential in the quantitative proteome analysis of biological samples.